The overall aim of this project is to elucidate the mechanism by which the target for Tat (transactivator) trans-activation TAR is produced. TAR is a RNA element encoded just downstream of the human immunodeficiency virus (HIV) transcriptional start site and, therefore, must be synthesized before Tat can stimulate gene expression. The investigator proposes to continue studies on the IST element and to study its role during HIV infection. The applicant first will try to localize IST precisely by analyzing the effects of mutations in the -5 to +82 HIV region. Different mutant constructs will be transfected into HeLa, COS and Jurkat cells by electroporation. Effects on formation of short transcripts will be analyzed both at the level of steady-state and nascent RNA. With IST localized precisely, the investigator anticipates being able to characterize trans-acting factors responsible for IST activity by performing a combination of in vitro transcription experiments and DNA (or RNA) binding assays. To characterize the role of IST during HIV infection, the applicant proposes to construct HIV viruses with either a debilitated IST element, a debilitated Tat gene, or both. The investigator will attempt to generate cell lines containing mutant proviruses and to determine which one produces short transcripts and which can be trans-activated by Tat supplied exogenously. If a Tat-, but not a Tat-/IST-, provirus can be trans-activated by Tat supplied exogenously, it would raise the possibility that, in infected individuals, Tat from one cell can activate latent proviruses in other cells and that IST plays a key role in the trans-activation process.